HMCs were plated onto 96-well plates at a density of 103 cells/well. After attachment for 24 hr, the culture medium was replaced according to each group, and the cells were cultured for an additional 24, 48, 72, or 96 hr. The supernatants were discarded, and 200 μL of culture medium containing 0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well. After a 4-hr incubation period, the culture medium was carefully removed, and 100 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well. The cells were placed in an incubator and vortexed at a low speed for 2 min to fully dissolve the formazan crystals. The optical density (OD) of each well was measured at 490 nm using the EON microplate reader (BIOTEK, Winooski, Vermont, USA).
Eon microplate reader
Manufactured by Agilent Technologies
Sourced in United States, Switzerland
The Eon microplate reader is a versatile and reliable instrument designed for absorbance-based detection in microplate assays. It provides accurate and precise measurements for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Cck 8 kit, by Dojindo Laboratories (2 mentions)
Tryptic soy agar plates, by Carl Roth (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Prism 6, by GraphPad (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Etest strips, by bioMérieux (1 mentions)
Mtt assay, by Beyotime (1 mentions)
Cck 8, by Beyotime (1 mentions)
T5500, by Merck Group (1 mentions)
Trichloroacetic acid, by Avantor (1 mentions)
Cell counting kit 8 cck 8, by Vazyme (1 mentions)
Acarbose, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Synergy 2 multi mode, by Agilent Technologies (1 mentions)
Accuskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Platereader af2200, by Eppendorf (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amplex red hydrogen peroxide peroxidase assay kit, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Prism software, by GraphPad (1 mentions)
40 protocols using eon microplate reader
1
MTT Cell Viability Assay Protocol
2
Cell Proliferation Assessment via CCK-8
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Cell proliferation was measured using a CCK-8 kit (Dojindo, Kumamoto, Japan). Cells were incubated with 10 μl of CCK-8 at 37 °C for 1.5 h, and the absorbance at wavelengths of 450 and 630 nm were applied using a BioTek Eon Microplate Reader (BioTek, Washington, CA, USA).
3
Cell Viability Evaluation of Fuc–Gel–MTN–Biomineralized HAp Scaffolds
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To examine cell viability in the presence and absence of the Fuc–Gel–MTN–biomineralized HAp scaffolds, we plated cells at a density of 1 × 104 cells/well in 96-well Falcon polystyrene tissue culture plates. After allowing the cells to spread and attach for 3 h, we added Fuc–Gel–MTN–biomineralized HAp scaffolds at varying concentrations to the wells. We tested 20, 40, and 80 μg/mL concentrations of the scaffold. The growth of the cells was monitored over a period of 24, 48, and 96 h. To determine cell viability, we performed MTT assay. The absorbance at 570 nm was monitored at each time point using a BioTek Eon microplate reader. Triplicate experiments were run in all cases. The absorbance of media alone was used at the blank and was subtracted from all samples. Percent cell viability was calculated according to the formula [(O.D of cell plus scaffolds)/(O.D of cells alone)] × 100. The standard deviations were calculated. Statistically significant differences were then determined using student’s t-test.
4
Quantitative Growth Kinetics Analysis
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The WT KPPR1 and KPPR1ΔgltA strains were cultured overnight in LB broth, then diluted to a uniform OD600 of 0.01 in the culture medium of interest the following day. Amino acids were supplemented at 10 mM unless otherwise indicated, and carbon sources were supplemented at 5 mg/mL. Cultures were incubated at 37°C with aeration and OD600 readings were taken every 15 min using an Eon microplate reader with Gen5 software (Version 2.0, BioTek, Winooski, VT) for up to 24 hours. To simultaneously assess doubling time, growth rate, lag time, non-sigmoidal growth due to stress and bacterial density, area under the curve analysis was used to quantify differences in growth [37 (link),38 (link)] using Prism 6 (GraphPad Software, La Jolla, CA).
5
Quantifying Cellular Function in Forebrain
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The cellular function was assessed in forebrain homogenates by determining MTT reduction to formazan crystals [36 (link),37 (link)]. Briefly, the brain homogenate (125 µL) was mixed with 4 μL of the MTT (5 mg/mL) and then incubated for 15 min at 37 °C. Samples were then centrifuged at 17,000× g for 3 min and the pellets were suspended in 250 μL with acid-isopropanol. Optical density was determined using an Eon microplate reader (Biotek Instruments, Winooski, VT, USA) at a wavelength of 570 nm. Results are expressed as the percentage of MTT reduction considering the control as 100%.
6
Antibiotic Resistance Screening Protocols
7
Cell Division Rate Determination
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After synchronization, the rate of cell division was determined at 0, 24, 48, 72, 96, and 120 h of growth in Grace insect medium with 10% (v/v) FBS with a methyl thiazolyl tetrazolium (MTT) assay (C0009, Beyotime, China). The cells (100 μL, 1 × 105 cells/mL) were incubated for 4 h in 96-well plates at 26°C in the dark and additional 4 h at 37°C in the dark after adding 100 μL formazan. The absorbance at 570 nm was measured with an Eon microplate reader (BioTek, VT, United States). The measurement was repeated in five culture wells.
8
Colorimetric Assay for (Fgr)GaOx Activity
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The activity of FgrGaOx was measured by the chromogenic ABTS [2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid)] assay, originally described by Baron et al.,70 with modifications. The standard reaction mixture (final volume: 200 μL) contained 270 μg (31 μm , assuming purity) horseradish peroxidase (HRP, from horseradish, Sigma‐Aldrich, Germany), 2 mm ABTS, and 50 mm 2 a in 50 mm HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid) buffer (pH 7.5); reactions were initiated by adding 5 μL of appropriately diluted enzyme sample to obtain initial rates of reaction. Absorbance was measured at 420 nm at 30 °C for 20 min in an Eon microplate reader (BioTek, US). Activity values were calculated on the basis of the extinction coefficient (36 000 L mol−1 cm−1 at 420 nm).71 Each reaction was performed in triplicate, at minimum.
9
Quantification of Inflammatory Markers in Biological Samples
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Commercially available ELISA kits were used to assay endotoxin core antibody (EndoCab Human IgG; Hycult Biotech, Uden, Netherlands) and soluble CD14 (sCD14; Quantikine ELISA) in serum specimens, and myeloperoxidase (MPO) in stool specimens (Immundiagnostik, Bensheim, Germany) at three different time points. All these assays were carried out according to the manufacturers’ descriptions with some modifications, as stated previously [25 (link)]. Brifely, Plasma samples were diluted at a ratio of 1:1000 and 1:200 for measuring sCD14 and EndoCAb. The 1:200 was used to measure the MPO level in fecal extracts. We had run our samples with standards in a duplicate manner. If any standard values outside of the manufacturers’ descriptions were found, we repeated the corresponding samples or excluded those from our analysis. For ELISA procedures, Eon microplate reader was used (BioTek, USA), to measure absorbances, and Gen5 software was used to calculate concentrations of markers unless stated otherwise.
10
Cell Viability Assay with CCK-8
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Cells were suspended with 100 μl medium, seeded into the 96-well plate (2000 cells/well), and cultured for indicated times. 10 μl CCK-8 (Beyotime Biotechnology, Shanghai, China) was added into each well, followed by incubation in cell incubator for 2 h. The absorbance was read at 450 nm by the Eon™ Microplate Reader (BioTek, Whiting, VT, USA).
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